A versatile nuclei extraction protocol for single nucleus sequencing in non-model species–Optimization in various Atlantic salmon tissues

The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species.


ABSTRACT
Single cell RNA sequencing has rapidly become a standard tool for profiling transcriptomic diversity across thousands of cells (Linnarsson and Teichmann, 2016), and is now being applied to a large diversity of species and tissues.The main limitation of this technology is that it requires the isolation of live cells from fresh tissue, severely restricting its applicability.As a result, single nuclei RNA sequencing (snRNA-seq), which consists of sequencing the RNA of only the nuclei of cells rather than of the whole cell, has been commonly adopted since it allows samples to be stored for several months prior to processing while yielding comparable results to whole cell sequencing (Kulkarni, et al., 2019;Slyper et al. 2021).A critical challenge for snRNA-seq is the successful extraction of high quality nuclei.This has spurred the recent publication of a number of dissociation protocols for nuclei extraction (Drokhlyansky et al. 2020;Eraslan et al. 2021;Melms et al 2021), however, these have largely been optimized for model species such as humans, and more and more single nuclei is being adopted in non-model species.
Here we present a robust protocol that enables the extraction of nuclei from frozen tissue adapted from those shown to work in different tissue types, such as human skin (Drokhlyansky et al. 2020;Eraslan et al. 2021;Melms et al 2021).Our protocol has been used to successfully extract nuclei from an array of different Atlantic salmon (Salmo salar) tissues including liver, skin, fin, spleen, head kidney and gill as well as in other species such as sole (Solea solea) nose and gonad, rabbit (Oryctolagus cuniculus) nasal tissue and nurse shark (Ginglymostoma cirratum) spleen.We present the protocol as applied to fin and skin as these are particularly challenging tissues to work with given their toughness and the presence of hard tissue (e.g., scales and bones), connective tissue and fat deposits.We include notes throughout the protocol so that the reader can optimise it for a variety of tissue types.While the protocol has been optimised to work with the Chromium 10x platform, the most commonly used high throughput microfluidic device, but can be used successfully for the extraction of nuclei for other platforms and applications.
The aim of this protocol is to capture 7,000 nuclei per single-nuclei RNA sequencing library using the Chromium Single Cell 3' Reagent Kits v2 or v3 (10X Genomics).Given its utility for isolating nuclei from difficult to dissociate tissue types, we anticipate that this protocol will be broadly applicable for snRNA-seq of non-model organisms and unconventional tissue types.This step is critical .The tissue must be preserved as fast as possible for optimal results.In the absence of liquid nitrogen, samples can be frozen in dry ice.Samples can be stored at -80 °C for up to a year prior to use.Older samples might still yield viable nuclei but this would need to be tested.

REAGENTS REAGENTS
All reagents should be chilled on ice prior to use.All reagents should be chilled on ice prior to use.Make fresh and chill prior to use, add RNAnase inhibitor right before nuclear isolation.RNAase inhibitor amount can up upped if it's an RNAse Rich tissue, up to 500 U per ml instead, tissue spends very little time in this buffer and is chilled at all time, which is why the amount of RNAase inhibitor can be lower.
Make fresh and chill prior to use, add RNAase inhibitor right before nuclear isolation .Dilute the Tween from 10% in stock solution with nfH2O before making the buffer.
RNAnase inhibitor amount can be upped if it's an RNAase rich tissue up to 1000 U per ml instead, the nuclear isolation will happen in this buffer so its more critical in here.
protocols.io| https://dx.doi.org/10.17504/protocols.io.261genwm7g47/v2 ** can top this up this to 2% BSA if the cells are clumping or look degraded .RNAase inhibitor is the most critical in this step as the nuclei will be in this buffer the longest can use up to 1000 U per ml.

BEFORE START INSTRUCTIONS
Sampling and storage for nuclear isolation.Sampling and storage for nuclear isolation.
Animals must be appropriately euthanized and immediately processed.Approximately ~60 mg of tissue is placed in one clearly labelled cryotube and immediately flash frozen in liquid nitrogen.This step is critical This step is critical .The tissue must be preserved as fast as possible for optimal results.In the absence of liquid nitrogen, samples can be frozen in dry ice.Samples can be stored at -80 °C for up to a year prior to use.Older samples might still yield viable nuclei but this would need to be tested.
All reagents should be chilled on ice prior to use.All reagents should be chilled on ice prior to use.
Samples should be kept frozen on dry ice until immediately before nuclei isolation, and all sample-handling steps should be performed on ice.The centrifuge should be pre chilled at 4 °C .
All reagents are given for 2 nuclear isolations.All reagents are given for 2 nuclear isolations.Amounts of buffer especially those that contain RNase should be adjusted appropriately for each experiment prepared prior and RNase added immediately before use.
Before using this prep for library preparation do a trial run.Before using this prep for library preparation do a trial run.
Recommended to do a trial especially on a new tissue type to adjust different parameters without adding RNase.Once parameters are adjusted such as mincing times, filter size and dilution in to final buffer in order to get good quality nuclei.C .Liver nuclei to long in dissociation buffer, nuclear membrane started to degrade.Can still be sequenced but not ideal.Note when staining nuclei with trypan blue asses nuclear quality as soon as possible as the nuclei will quickly degrade when not on ice.

3.1
Add a further 1 mL of TST to the cell strainer immediately.

3.2
Add 3 mL of freshly prepared ST buffer to the lysate.

3.3
Add the 5 mL of lysate to a marked 15 ml falcon tube (Corning) on ice.
LoBind Tubes, 1.5ml, PCR clean Cryotube 6-well tissue culture plate (Stem Cell Technologies) Falcon tubes 15 ml (Corning) INCYTO C-Chip™ Disposable Hemacytometers VWR International VWR International Catalog Catalog # #82030-468 82030-468 SAMPLING AND STORAGE FOR NUCLEAR ISOLATION SAMPLING AND STORAGE FOR NUCLEAR ISOLATION Animals must be appropriately euthanized and immediately processed.Approximately ~60 mg of salmonid tissue is placed in one clearly labelled cryotube and immediately flash frozen in liquid nitrogen.This step is critical Tris-HCl pH 7.5: UltraPure™ 1 M Tris-HCI Buffer, pH 7.5 Thermo Fisher Thermo Fisher Catalog # Catalog #15567027 15567027 CaCl2: protocols.io| https://dx.doi.org/10.17504/protocols.io.261genwm7g47/v2Calcium chloride 1 M in aqueous solution VWR International VWR It is important to use the correct RNAse inhibitor as it can negatively affect library prep, check with the sequencing platform before using another type of RNAse.Do not add RNAse until right before nuclear extraction.RNAse inhibitor does not need to be used to test nuclear extractions, but it should added for sequencing runs.1X ST buffer solution (ST) ST) -10 mL : Dilute 2x ST in ultrapure nuclease-free water (1